Spectrum of chloroperoxidase compound I

Palcic, Monica M.; Rutter, Rick; Araiso, Tsunehisa; Hager, Lowell P.; Dunford, H. Brian

doi:10.1016/0006-291x(80)90535-5

Cited by 136 publications

(103 citation statements)

References 8 publications

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“…The preparation of compound I Peracetic acid was chosen as the oxidant because it is an excellent oxidant for compound I formation and it is a poor substrate for the catalase activity of chloroperoxidase, which causes compound I disappearance [5, 61. It was shown at pH 4.6 that a sixfold excess of peracetic acid is required to obtain 100% compound I [5]. When chloroperoxidase is mixed with an equimolar amount of peracetic acid in the stopped-flow apparatus, the absorbance at 400 nm decreases as compound 1 is formed and subsequently increases as ?ompound I decays.…”

Section: Resultsmentioning

confidence: 99%

Kinetics of the oxidation of ascorbic acid, ferrocyanide and p‐phenolsulfonic acid by chloroperoxidase compounds I and II

Lambeir¹,

Dunford²,

Pickard³

1987

European Journal of Biochemistry

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For the first time elementary reactions involving chloroperoxidase compounds I and I1 have been investigated. A multi-mixing stopped-flow apparatus was used to study the kinetics of the reactions of compounds I and 11 with ascorbic acid, ferrocyanide and p-phenolsulfonic acid. The second-order rate constants of the reactions of both compounds with all three substrates were determined between pH 3 and pH 7. In all cases the rate constants decrease with increasing pH. The reactions of p-phenolsulfonic acid are influenced by a catalytically important group on both compounds I and I1 with a pK, of 3.7 f 0.2. With ascorbic acid and ferrocyanide as substrates, a decrease in rate was observed upon ionization of the substrate. Comparisons with horseradish peroxidase show that chloroperoxidase is a much less efficient peroxidatic enzyme. The kinetic data were used to calculate the percentage composition of the mixture of chloroperoxidase species which contribute to the spectra measured during the turnover with ascorbate as substrate.Chloroperoxidase derives its name from its ability to use chloride ion as a substrate in halogenation reactions [l, 21. Chloroperoxidase also catalyzes the dismutation of hydrogen peroxide and the oxidation of ethanol to acetaldehyde, reactions typical of catalase, and the peroxidation of organic compounds with a mechanism similar to that of the plant peroxidases [3]. The first step in the mechanism of all the reactions is the oxidation of the native enzyme by hydrogen peroxide, alkyl peroxides or peracids to form compound I [4 -61. The kinetics of compound I formation from hydrogen peroxide or peracetic acid have been studied in some detail and the rate constant (kl, app) was reported to be independent of the pH between pH 3 and pH 7 [5-71. Compound I of chloroperoxidase has a formal oxidation state of + 5 and it has the spectral properties of an oxyferryl n cation radical [8, 91, as has compound I of horseradish peroxidase. In the halogenation and catalase reactions compound I receives two electrons from the substrate (halide ion, hydrogen peroxide or ethanol) to form a ferric-halogenium intermediate or native enzyme [4,6,7,10, 111. Alternatively compound I can react with a one-electron donor to form compound 11, which returns to the native enzyme by reacting with a second substrate molecule [3, 41.

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Section: Resultsmentioning

confidence: 99%

Kinetics of the oxidation of ascorbic acid, ferrocyanide and p‐phenolsulfonic acid by chloroperoxidase compounds I and II

Lambeir¹,

Dunford²,

Pickard³

1987

European Journal of Biochemistry

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“…Therefore a tyrosine radical, similar to what we have observed, cannot be confirmed by their experiments. There is, however, a discrepancy in the characterization of the iron-oxo intermediate in P450cam which arises from comparison with the stopped-flow studies by Egawa et al (6) who mixed substrate-free P450cam with mCPBA and found in the same time interval (6 -8 ms) an electronic absorption spectrum that is similar to that seen for the Fe(IV)ϭO porphyrin--cation radical of CPO (71). There are two differences between the stopped-flow and freezequench experiments.…”

Section: Implications For the Catalytic Mechanismmentioning

confidence: 91%

Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

Schünemann

Lendzian²,

Jung

et al. 2004

Journal of Biological Chemistry

108

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“…Dioxygen binding yields the oxy-ferrous adduct, a ferrous-O 2 /ferric superoxide resonance hybrid (4a 7 4b). CO binding to 3 generates the ferrous-CO derivative (5). Addition of the second electron from reduced Pdx has been proposed to yield a ferric peroxo species (6a), protonation of which gives the hydroperoxo state (6b).…”

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confidence: 99%

Direct Observation of a Novel Perturbed Oxyferrous Catalytic Intermediate during Reduced Putidaredoxin-initiated Turnover of Cytochrome P-450-CAM

Glascock

Ballou

Dawson

2005

Journal of Biological Chemistry

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The single turnover of (1R)(؉)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3°C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high and low spin Fe(III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, ϳ140 and ϳ85 s ؊1, were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM⅐Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substratefree P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron.The cytochrome P-450 family of heme-containing mono-oxygenases is involved in the metabolism of xenobiotics and in the production of physiologically important molecules (1). A defining characteristic of the P-450 family is the proximal thiolate-ligated heme. P450-CAM (P450-CAM, CYP101) 3 from Pseudomonas putida catalyzes the hydroxylation of (1R)(ϩ)-camphor to form (1R)(ϩ)-5-exo-hydroxycamphor (Reaction 1). The electrons required for the reaction flow from NADH to the flavoprotein, putidaredoxin reductase, then to the iron-sulfur (2Fe/2S) protein, putidaredoxin (Pdx), and finally to P450-CAM. In the widely quoted P450 reaction cycle shown in Fig. 1 (1), substrate binding converts the ferric low spin resting state (1) to the ferric high spin form (2). Electron transfer from reduced Pdx gives high spin deoxyferrous P-450 (3). Dioxygen binding yields the oxy-ferrous adduct, a ferrous-O 2 /ferric superoxide resonance hybrid (4a 7 4b). CO binding to 3 generates the ferrous-CO derivative (5). Addition of the second electron from reduced Pdx has been proposed to yield a ferric peroxo species (6a), protonation of which gives the hydroperoxo state (6b). Protonation of the distal oxygen produces water and compound I, a ferryl porphyrin -cation radical (7). This highly oxidizing intermediate abstracts a hydrogen...

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Spectrum of chloroperoxidase compound I

Cited by 136 publications

References 8 publications

Kinetics of the oxidation of ascorbic acid, ferrocyanide and p‐phenolsulfonic acid by chloroperoxidase compounds I and II

Kinetics of the oxidation of ascorbic acid, ferrocyanide and p‐phenolsulfonic acid by chloroperoxidase compounds I and II

Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

Direct Observation of a Novel Perturbed Oxyferrous Catalytic Intermediate during Reduced Putidaredoxin-initiated Turnover of Cytochrome P-450-CAM

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